The objective of this proposal is to begin addressing the role of protein Ser/Thr/Tyr phosphorylation in regulating chlamydial physiology and pathogenesis through initial elucidation of the protein kinase/phosphatase networks. Protein Ser/Thr/Tyr phosphorylation is increasingly recognized as a widely- employed mechanism for regulating bacterial processes crucial for growth and survival (and therefore pathogenesis) via reversible alteration of protein function. Chlamydia spp. undergo a unique biphasic developmental cycle transitioning between the environmentally stable EB and the replicative, intracellular RB that would appear to require extensive regulation of protein synthesis and function. Perplexingly, <5% of their genomes are reserved for canonical transcriptional regulators. Furthermore, early differentiation of EBs into RBs occurs under conditions where DNA is occluded by histone-like proteins and only low levels of RNA are present. These biological problems suggest the use of non-classical solutions for regulating pathway functionality in Chlamydia spp. Consequently, we hypothesize that Chlamydia spp. utilize global protein phosphorylation as a mechanism to respond to stressors and regulate differentiation. Consistent with this hypothesis, mapping of the EB/RB phosphoproteomes of C. caviae using 2D gel electrophoresis, phosphoprotein staining, and MALDI-TOF-TOF analysis identified 42 stage-specific phosphorylated proteins in EBs and RBs (98% were pan-chlamydial proteins). In addition, 1D gel electrophoresis phosphoprotein staining analysis of Chlamydia spp. further supports the presence of abundant levels of phosphoproteins across Chlamydia. The work proposed in this R15 study will further address our hypothesis using a systematic, bottom-up approach that focuses on building and validating kinase/substrate and phosphatase/substrate interactomes focusing on Chlamydia trachomatis. In Aim 1, we will delineate the Pkn1 and PknD kinase/substrate network using multiple methods to detect protein-protein interactions. Partnering will be confirmed using in vitro kinase assays and phosphorylation sites also will be determined for select substrates. We will seek to validate the functionality of CTL0511, a predicted protein phosphatase, in Aim 2. CTL0511 substrates will then be identified using in vitro phosphatase assays and various protein-protein interaction methods. Finally, in Aim 3, we will further define the interacting partners comprising the phosphorylation- regulated chlamydial Partner Switching Mechanism. Collectively, these studies will fill a gap in our understanding of chlamydial development, provide future research directions, identify novel targets for therapeutics and anti-infectives, and illuminate the broader consequences of protein phosphorylation on bacterial physiology and pathogenicity.